Biologically active molecules, particularly based on pna and sirna, method for the cell-specific activation thereof, and application kit to be administered

ABSTRACT

Biologically active molecules are inactivated for selective activation by target cells by being covalently bonded to one or more peptides each of which has one or more specific amino acid sequences that are selected in respect of enzymes cell-specific for target cells. The bonds, which are broken exclusively by the enzymes cell-specific for the target cells in order to biologically activate the molecules, allow the molecules to remain biologically inactive in cells other than the target cells. The molecules are used for influencing gene expression of preferably sick and infected organs or cells, for example.

BACKGROUND OF THE INVENTION

The invention relates to special biologically active molecules,particularly based on peptide nucleic acids (PNA) and short interferingRNA (siRNA), a method for their transfection into a target cell andcell-specific activation in this cell or directly before theirtransfection, and an application kit to be administered in combinationwith a transfection system. Said biologically active molecules interactwith the mRNA of the target gene and in the case of siRNA they formtogether with specific endoribonucleases an RNA protein complex namedRISC (RNA induced silencing complex). The RISC complex bonds to thetarget mRNA and endonucleases restrict the target mRNA. In this way,gene expression is suppressed and consequently the formation of targetproteins is inhibited. If activated PNA molecules are used, thetranslation will be prevented due to the bonding to the target mRNA.

The cell-specifically activatable, biologically active molecules can beused, for example, for combating abnormal cells and inhibiting theirgrowth, particularly in tumor treatment, treatment of virus infections,and age-specific treatments for example. Generally, thecell-specifically activatable, biologically active molecules can be usedfor the modulation of gene expression of the target cells. Thismodulation does not only allow reduction of the gene expression but alsoincrease thereof by achieving a reduction of the expression of thenegative regulators of the target gene by means of the biologicallyactive molecules.

The inhibition of gene expression by introducing short (19-23 bp),double-stranded RNA molecules (siRNA) or PNA molecules in eukaryoticcells, which is specific for a sequence segment of the mRNA of a targetgene, has already been described (Elbashir S M et al.: Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells,Nature, 2001 May 24, 411(6836), 494-8; Liu Y et al.: Efficient andisoform-selective inhibition of cellular gene expression by peptidenucleic acids, Biochemistry, 2004 Feb. 24, 43(7), 1921-7; U.S. Pat. No.5,898,031; U.S. Pat. No. 7,056,704).

The use of such molecules does not prevent the reading of a gene and thegeneration of an mRNA, but in the case of siRNA it initiates anendogenous mechanism that degrades the target mRNA. Finally, as writtenabove, the formation of a specific protein is suppressed withoutimpairing the expression of further genes (post-transcriptional genesilencing).

To suppress the expression of a gene, the siRNA and PNA molecules can bedirectly introduced into the cell, particularly via transfectionreagents and electroporation (Zhang M et al.: Downregulation enhancedgreen fluorescence protein gene expression by RNA interference inmammalian cells, RNA Biol. 2004 May, 1(1), 74-7; Gilmore IR et al.:Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22,Curr Drug Deliv. 2006 April, 3(2), 147-5; U.S. Pat. No. 6,506,559). Thedisadvantage of this method is the relative instability of the siRNA butit can be reduced by chemical modifications (U.S. Pat. No. 6,107,094).

A particular problem for the therapeutic application of biologicallyactive molecules is an application in vivo. Methods for stabilizing thesiRNA have been developed for such an application in order to reduce thedegradation (Morrissey et. al.: Chemical Modifications of SyntheticsiRNA, Pharmaceutical Discovery, May 1, 2005), and transfection reagentshave been engineered, for example nanoparticles, in vivo-jetPEI™(Polyplus), that introduce the siRNA into the cells in vivo, too(Vemejoul et al.: Antitumor effect of in vivo somatostatin receptorsubtype 2 gene transfer in primary and metastatic pancreatic cancermodels, Cancer Research 62, 2002, 6124-31; Urban-Klein B, Werth S,Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting throughsystemic application of polyethylenimine (PEI)-complexed siRNA in vivo,Gene Ther 12(5), 2005, 461-6.).

Furthermore, methods have been evolved to increasingly transfect siRNAinto cells of a target tissue in vivo (Ikeda et. al.: Ligand-TargetedDelivery of Therapeutic siRNA, Pharmaceutical Research, Vol. 23, No. 8,August 2006).

However, the administration of biologically active substances in vivo isoften problematic due to the systemic effect. The selective introductionof these substances into target cells is not sufficiently specific. Thisdisadvantage is particularly important for siRNA and PNA molecules thatshall selectively act and shall have this selective effect only in thetarget cells. The cell specificity achieved by transfection reagentsthat are provided with a tissue or cell marker (e.g.antibody/antigene-marked nanoparticles, TAT protein flanking and others)is not sufficiently high. Wrong transfection is the result.

Moreover, a method is known that deactivates the biological effect ofsiRNA molecules by bonding fluorochromes and bringing back saidmolecules to their active state by exposing them to light of a definedwave length (QN Nguyen et al.: Light controllable siRNAs regulate genesuppression and phenotypes in cells, Biochim Biophys Acta, 2006). Thisactivation is initiated from the outside and is not cell-specific in anyway. After their activation said siRNA molecules have consequently anundesired effect in all the other transfected cells, too and not only inthe target cells as intended.

Furthermore, it is also difficult to use this mechanism for in vivoapplications.

SUMMARY OF THE INVENTION

The aim of the invention is to produce biologically active substancesthat can be transfected into a target cell both in vitro and in vivo andinhibit gene expression exclusively here without influencing thesubstance-specific expression of the target gene and thus the formationof protein in other cells of the organism.

For the treatment of tumors, which has been mentioned as a possibleapplication, this method shall selectively suppress the expression ofthe target gene and thus the protein formation in tumor cells withoutinfluencing healthy cells—which can also be reached by the activesubstances—and their continued existence.

Said aim is achieved by a covalent bond of the biologically activemolecules, particularly PNA and siRNA, to one or more peptides each ofwhich has at least one specific amino acid sequence that is selected inrespect of enzymes typical of the target cells and is significant forthe covalent bond and its breaking. Said covalent bond deactivates thebiologically active molecules. Therefore, a specific gene expression isnot inhibited after the transfection in cells as long as even only oneof the bonded peptide chains remains at the PNA or siRNA molecules dueto the non-existence of the corresponding enzyme typical of the targetcell. By an appropriate transfection system, for example nanoparticlesor coat molecules such as liposomes, the deactivated substance moleculescan be transfected into the target cells. Here, said deactivatingcovalent bonds are broken in a cell-typical manner by the one or morecell-specific enzymes relevant for the amino acid sequences of the oneor more coupling peptides. Thus, the biological strength of the moleculethat is now within the target cell and separated from peptides isactivated. As a result, the molecule bonds to the specific mRNA of thetarget cell and inhibits the gene expression in this special cell in themanner as such.

Unlike in the predefined target cells, in all other cells of theorganism that can also be reached by the molecules said molecules remaininactive because the covalent bonds between the biologically activemolecule, in particular PNA and siRNA, and the one or more peptidespersist completely (no peptide bond has been broken) or partly (not allpeptide bonds have been broken) because of the non-existence of thetarget enzyme or enzymes typical of the target cell. Due to thecontinuing covalent peptide bond the biologically active molecule doesnot form a bond with the mRNA of this cell and, if siRNA is used, a RISCwill not be initiated.

Even though the inventive molecule constructs that are to be transfectedin their unbound form, for example in a tumor treatment, do not only getto or at a tumor target cell but can also reach healthy cells (what ishardly to be avoided in practice) said molecule will be activatedselectively and exclusively in or at the tumor target cells by thecell-specific enzyme present here and the substance-related expressionof the target gene will be suppressed. Although the molecule constructsare present in the not sick (or for the intended biological effect notpurposive) cells gene expression and thus protein formation for thecontinuing existence of the healthy cells remain unaffected from thissubstance because said molecule constructs are permanently inactive.

It is also possible that even directly before the transfection of theone or more covalent bonds into the target cell each of said bonds isactivated partly or completely (referring to the total number ofcovalent peptide bonds) by one or more cell-specific enzymes being onthe surface or in the vicinity of the target cell.

For example, several peptide chains with amino acid sequencesindividually selected for the different enzymes of the target cell couldexist and each of one or more bonds is already broken directly on thesurface or in the vicinity of the target cell.

It is also conceivable that the biologically active molecules in theirinactive state, which is caused by said peptide bonds, get to the targetcells or the target tissue and there they are completely activatedbefore their transfection into the target cell, particularly if theycannot reach other cells in this part of the organism.

Thanks to the proposed highly selective effect of the molecules as aresult of the target-cell-enzyme-specific inactivity/activity thebiologically inactive molecule constructs to be transfected can beadministered systemically, if an appropriate peptide bond (with definedamino acid combinations) is formed.

The molecule constructs can be bonded in or to the target cells toensure a better transport or to other substances (for examplenanoparticles as a carrier system) to ensure the stability of saidmolecule constructs.

Moreover, well-known mechanisms that increase the tissue or cellselectivity (for example ligand/antibody/antigene-marked nanoparticles,TAT protein flanking) can be used to increase the transfection rate. Thecombination with these known mechanisms complements the describedinvention and leads to a reduction of the amount of the inventivesubstance due to the pre-selection of the target cells. This advantageresults from the fact that the active substance mostly reaches cells intissue with a good blood flow when it is transfected in vivo into theblood circulation. The proposed molecules with the inventivebiologically deactivating bond to one or more peptides can be very wellused in combination with generally known transfection systems, such asnanoparticles with or without ligand/antibody/antigene marking, withcoating nanoparticles or lipids and lipid-based transfection methods.Depending on the intended application, such transfection systemsthemselves already decrease organism-caused wrong transfections andtherefore, considered in relation, an increased number of cells istransfected into the target tissue so that the amount of substance to beused can be limited to a minimum.

If this invention is applied, the wrong transfections cannot be actuallyinhibited but the wrongly transfected molecules are not biologicallyactive in the cells other than the target cells, even though they arestill undesired in them. The inactive form of the molecules also remainsunchanged despite the inventive molecule activation in or at the targetcells so that the biological effect of the molecules is only active inthe target cells and a highly cell-selective modulation of geneexpression is reached, unlike in different mechanisms known so far.

The invention is not limited to applications for treating tumors and,for example, cells infected by viruses (e.g. HIV). The inventivemolecule constructs can be used for all clinical syndromes that arebased on an increased or reduced gene expression in order to treat thedisease itself or its symptoms. Furthermore, age- ordevelopment-specific changes in the gene expression pattern of cells canbe treated. Additionally, the invention can be used for all applicationsin vitro or in vivo that aim at the selective change in gene expressionin specific cells.

It is advantageous to use an application kit that provides thebiologically active molecules with the bonded peptides that have—asproposed—amino acid sequences selected in respect of enzymes typical ofthe target cells and an appropriate transfection system as well as otheradditives. The application kit contains all required substances and amanual to instruct the user about the intended operation.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following, examples explain the invention in detail in schematicdrawings.

They show:

FIG. 1: general structure of the inventive cell-specific activatablemolecule constructs (deactivated state; due to a changed space structureof the molecules the formation of the RISC is suppressed in example a)and in example b) the bond to the mRNA is prevented)

a) example for siRNA with two bonded peptides

b) example for PNA with one bonded peptide

FIG. 2: example for a siRNA molecule construct bonded to a transfectionsystem for the transfection into the target cells

a) biologically inactive molecule construct for the transfection intothe target cell

b) bond breakage by the enzyme typical of the target cell

c) molecule biologically activated in or at the target cell with brokenbond between the siRNA and the peptide

FIG. 3: siRNA molecule construct with two peptide bonds and onetransfection system for the transfection into the target cells

a) molecule construct with peptide bonds

b) breakage of the peptide bonds by different enzymes of the target cell

FIG. 4: representation of an exemplary bond between a peptide and ansiRNA; in this case a possible enzyme for decomposing the peptide bondis Caspase-4

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows the general structure of the inventive cell-specificactivatable molecule constructs in a deactivated state as an example forsiRNA (FIG. 1 a) and for PNA (FIG. 1 b).

In FIG. 1 a, a siRNA 1 as a biologically active molecule is bonded totwo peptides 2, 3. Due to these bonds on both sides the siRNA 1 isbiologically inactive and is transfected into a target cell (not shownfor the sake of clarity).

FIG. 1 b demonstrates how a PNA 4 instead of the siRNA 1 is bonded as abiologically active molecule to the peptide 2 and is thus alsobiologically deactivated for the transfection.

FIG. 2 shows a possible construct that could be used for thetransfection of the molecule (siRNA 1), which is biologicallydeactivated by the peptide (peptide 2), into one target cell. And (FIG.2 a) to a nanoparticle 5 antibodies 6 could be bonded for thesemi-selective bonding to target cells and polyethylene glycol chains(PEG) 7 for anchoring the peptide 2 and the siRNA 1.

Furthermore, the bonding between the siRNA 1 and the deactivatingpeptide 2 is shown as an restricting site 8 for the breakage by aselectively restricting enzyme 9 typical of the target cell (FIG. 2).This enzyme 9, which is only present in or at the aforesaid (not shown)target cell, breaks the peptide bond of the siRNA 1 at the restrictingsite 8 due to the specific amino acid sequence (FIG. 2 c). The molecule(siRNA 1) that is again biologically active now because of the brokenpeptide bond and the residual construct consisting of the nanoparticle5, the antibody 6, the polyethylene glycol chains (PEG) 7 and thepeptide removed from the siRNA 1 are consequently separated.

According to the invention the peptide bond of the siRNA 1 at therestricting site 8 still exists in or at other cells of the organismthat do not belong to the intended target cells and that are alsoreached by the biologically inactive molecule construct (see FIGS. 2 aand 2 b) by transfection and in or at which the enzyme 9 typical of thetarget cell is not present. The siRNA 1 as a biologically activemolecule continues to be inactive (cp. FIG. 2 a). The biological effectof the siRNA 1 desired in the target cells is suppressed in other cellsby the unbroken restricting site 8.

FIG. 3 a shows an extension of the construct of FIG. 2. Here, a peptide2, 3 is also bonded to different points of the siRNA 1 in order todeactivate its biological effect (cp. FIG. 1 a). The different aminoacid sequences in the peptides 2, 3 are selected so that two specificenzymes (exclusively) present in or at the target cell can break thepeptide bonds of these restricting sites 8 or 8′. If even only one ofthe two peptides 2, 3 is not present at a cell that is also reached bythe construct according to FIG. 3 a (e.g. after a wrong transfection)but does not belong to the target cells, at least one of the two peptidebonds persists at the restricting site 8 or 8′ due to the missing of theequivalent enzyme 9, 10 typical of the target cell. The siRNA 1 willeven continue to be inactive, if only a single peptide bond stillexists. Only at the target cell in or at which the enzymes 9, 10 breakthe two restricting sites 8, 8′ (sketched out in FIG. 3 b) by the abovementioned defined amino acid sequences of the peptides 2, 3, the siRNA 1will be separated from the rest of the molecule construct (cp. also FIG.2 c). Thus, the siRNA 1 as a biologically active molecule can onlydevelop its intended effect by the transfection into this target cell.

FIG. 4 shows a possible bond between the siRNA 1 and one peptide withone possible specific amino acid sequence 11 (amino acid sequence is-L-E-V-D-) demonstrated for a selectively separating enzyme Caspase-4that is present in one target cell. Said enzyme would break the bond forthe activation of the siRNA 1 at the separation point for Caspase-4 thatis symbolized by the arrow and indicated in words.

In the illustrated example, a molecule rest would remain at the siRNA 1after the aforesaid separation by the enzyme Caspase-4 but this wouldnot impair the biological activity of siRNA 1.

The invention is not limited to the just indicated amino acid sequence(-L-E-V-D-) shown in FIG. 4 with respect to the breakage of the peptidebond at the siRNA 1 by means of the enzyme Caspase-4 typical of thetarget cell. The following table contains a list of examples of otheramino acid sequences of the peptide that can be used for the proposedapplication for special target cell enzymes:

Amino acid sequence Target cell enzyme of the peptide Matrixmetalloproteinase-1 -Pro-Leu-Ala-Leu-Trp-Ala-Arg- Matrixmetalloproteinases-2,7 -Pro-Leu-Gly-Leu-Dpa-Ala-Arg- Matrixmetalloproteinases-2,9 -Pro-Leu-Gly-Met-Trp-Ser-Arg- Matrixmetalloproteinases-3,1,2,9 -Arg-Pro-Lys-Pro-Tyr-Ala-Nval- Trp-Met-Lys-Cathepsin-S -Phe-Arg-Phe(p-nitro)- Cathepsin-G -Ala-Ala-Phe- Cathepsin-D-Arg-Gly-Phe-Phe-Leu- Angiotensin converting enzyme -Gly-His-Leu- or-Phe-Gly-Gly- or -Gly-p-Nitro-Phe-Pro

The four target cell enzymes listed first in the above table can also bepresent on the surface of the target cell or in its vicinity. In such acase and for such an application of the biologically active moleculessaid four target enzymes would be able to break the correspondingpeptide bond by the inventively selected amino acid sequence evendirectly before the transfection into the target cell. This applicationwill be particularly possible, for example for the transfection into atarget cell complex, if the biologically active molecules, which havereached the range of the target cell in their inactive state, cannot getto other (non-desired) cells in this part of the organism after theircomplete or partial activation already performed outside the targetcell.

It is advantageous to use an application kit that provides the requiredbiologically active molecules with the bonded peptides that have—asproposed—amino acid sequences selected in respect of enzymes typical ofthe target cells. The application kit should contain all necessarysubstances in ampoules, purposefully also a selection of appropriatetransaction systems (such as nanoparticles, ligands and polyethyleneglycol) as well as one or several probes or syringes with hollow needlesfor the injection of the mixture of the contents of the ampoules intothe medium of the target cells. The user can prepare and applyappropriate application mixtures and use them as described in a suppliedinstruction manual that contains a list of the selectable peptide aminoacid sequences and the corresponding enzymes of the target cells (cp.Table above).

It would be favorable to prepare such an application kit specificallyfor selected target cells and target genes in accordance with the typeof application (in vitro or in vivo).

1. Biologically active molecules, which are biologically inactivated fortransfection into a target cell to inhibit gene expression therein,after biological activation, by bonding to mRNA wherein, for theirbiologically deactivated state outside the target cell, the biologicallyactive molecules have at least one covalent bond with at least onepeptide having one or more amino acid sequences selected for one or moreenzymes cell-specific for the target cell and each of which enzymescontributes to breaking the covalent bond, and said bond can only bebroken by said one or more enzymes cell-specific for the target cell. 2.Inactivated biologically active molecules according to claim 1, whereinthe at least one peptide has an amino acid sequence -L-E-V-D- forbreaking the bond by the cell-specific enzyme Caspase-4.
 3. Inactivatedbiologically active molecules according to claim 1, wherein the at leastone peptide contains an amino acid sequence-Pro-Leu-Ala-Leu-Trp-Ala-Arg- for breaking the bond by the cell-specificenzyme matrix metalloproteinase-1.
 4. Inactivated biologically activemolecules according to claim 1, wherein the at least one peptidecontains an amino acid sequence -Pro-Leu-Gly-Leu-Dpa-Ala-Arg- forbreaking the bond by the cell-specific enzymes matrixmetalloproteinases-2,7.
 5. Inactivated biologically active moleculesaccording to claim 1, wherein the at least one peptide contains an aminoacid sequence -Pro-Leu-Gly-Met-Trp-Ser-Arg- for breaking the bond by thecell-specific enzymes matrix metalloproteinases-2,9.
 6. Inactivatedbiologically active molecules according to claim 1, wherein the at leastone peptide contains an amino acid sequence-Arg-Pro-Lys-Pro-Tyr-Ala-Nval-Trp-Met-Lys- for breaking the bond by thecell-specific enzymes matrix metalloproteinases-3,1,2,9.
 7. Inactivatedbiologically active molecules according to claim 1, wherein the at leastone peptide contains an amino acid sequence -Phe-Arg-Phe(p-nitro)- forbreaking the bond by the cell-specific enzyme Cathepsin-S. 8.Inactivated biologically active molecules according to claim 1, whereinthe at least one peptide contains an amino acid sequence -Ala-Ala-Phe-for breaking the bond by the cell-specific enzyme Cathepsin-G. 9.Inactivated biologically active molecules according to claim 1, whereinthe at least one peptide contains an amino acid sequence-Arg-Gly-Phe-Phe-Leu- for breaking the bond by the cell-specific enzymeCathepsin-D.
 10. Inactivated biologically active molecules according toclaim 1, wherein the at least one peptide contains an amino acidsequence -Gly-His-Leu- for breaking the bond by the cell-specific enzymeangiotensin-converting enzyme.
 11. Inactivated biologically activemolecules according to claim 1, wherein the at least one peptidecontains an amino acid sequence -Phe-Gly-Gly- for breaking the bond bythe cell-specific enzyme angiotensin-converting enzyme.
 12. Inactivatedbiologically active molecules according to claim 1, wherein the at leastone peptide contains an amino acid sequence -Gly-p-Nitro-Phe-Pro- forbreaking the bond by the cell-specific enzyme angiotensin-convertingenzyme.
 13. Method for cell-specific activation of biologically activemolecules in or at a target cell, in which the biologically activemolecules are to be transfected in their biologically deactivated stateinto a target cell to inhibit gene expression therein, after theirbiological activation, by bonding to mRNA comprising, for the biologicalinactivation of the biologically active molecules outside the targetcell or before the transfection, covalently bonding said molecules toone or more peptides each having at least one amino acid sequence thatis selected for one or more enzymes cell-specific for the target celland each of which enzymes contributes to breaking of the bond, thecovalent bond of the one or more peptides to the biologically activemolecule being broken by each of the enzymes cell-specific for thetarget cell thereby to activate the biological effect of the molecules.14. Method according to claim 13, further comprising transfecting theinactivated biologically active molecules into the target cells whereinthe inactivated molecules are activated by said one or morecell-specific enzymes therein.
 15. Method according to claim 14, whereinthe transfection is via a carrier.
 16. Method according to claim 14,wherein the carrier comprises nanoparticles or lipids, with which theselectively activatable biologically active molecules are covered fortheir transfection.
 17. Method according to claim 14, wherein thecarrier comprises lipids.
 18. Method according to claim 14, wherein thetransfection is by way of a TAT protein flanking.
 19. (canceled) 20.Method according to claim 13, wherein the inactivated biologicallyactive molecules are partly or completely, with respect to the totalnumber of the covalent peptide bonds, activated before the transfectioninto the target cell by one or more of the cell-specific enzymes beingpresent on the surface of or vicinity of the proximate to target cell.21. Method according to claim 13, further comprising coupling theinactivated biologically active molecules to one or more markersubstances.
 22. Kit for administering the inactivated biologicallyactive molecules of claim 1 comprising at least one of a first ampoulecontaining, as the biolgoically active molecules, a siRNA that iscovalently bonded, for the purpose of deactivation, to said at least onepeptide or a second ampoule that contains a PNA that is covalentlybonded, for the purpose of deactivation, to said at least one peptide,at least one third ampoule that contains a transfection medium, diluentand reaction buffers for the contents of the first, second and thirdampoules, at least one probe or syringe having a hollow needle forinjection of a mixture of contents of the ampoules into a mediumcontaining the target cells, and an instruction manual containing a listof peptide amino sequences selectable for the correspondingcell-specific enzymes of the target cell.
 23. Kit according to claim 22,further comprising at least one substance for semi-selective bonding ofthe biologically active molecules to the target cells.
 24. Kit accordingto claim 22, further comprising at least one substance for anchoring thepeptide and the biologically active molecules.
 25. Kit according toclaim 22, further comprising at least one substance for the transfectionof the biologically active molecules into the target cell. 26.(canceled)
 27. (canceled)
 28. Inactivated biologically active moleculesaccording to claim 1, comprising a PNA or siRNA having said at least onecovalent bond with said at least one peptide.
 29. Inactivatedbiologically active molecules according to claim 1, comprising siRNAhaving said at least one covalent bond with said at least one peptide,the bonding of siRNA to mRNA to occur through a RISC complex.
 30. Methodaccording to claim 13, wherein the biologically active moleculescomprise a PNA or siRNA, the bonding of siRNA to mRNA to occur through aRISC complex.
 31. Method according to claim 15, wherein the carriercomprises nanoparticles.
 32. Method according to claim 15, wherein thecarrier comprises nanoparticles with at least one of ligand, antibody oranti-gene marking.
 33. Method according to claim 20, wherein thecell-specific enzymes comprise at least one of matrixmetalloproteinase-1, matrix metalloproteinases-2, matrixmetalloproteinases-3, matrix metalloproteinases-7, or matrixmetalloproteinases-9.
 34. Kit according to claim 23, wherein thesubstance comprises antibodies.
 35. Kit according to claim 24, whereinthe substance includes polyethylene glycol chains.